Stromal cell production of signaling molecules is involved in neoplastic transformation and can directly regulate growth and survival of established cancer cells. Fibroblast activation protein alpha (FAP) is selectively produced by reactive stromal cells present within sites of epithelial cancers but is not expressed within stroma of any other adult tissues. FAP is a membrane bound serine protease with dipeptidyl peptidase, gelatinase and collagenase enzymatic activities. Therefore, the objective of this proposal is to determine if selective elimination of tumor associated stromal cells by a FAP activated prodrug would be effective, targeted treatment for cancer. To achieve this objective the following Specific Aims are proposed: (1) to define selective peptide substrates for the proteolytic activity FAP;(2) to synthesize FAP-cleavable prodrugs by coupling the FAP peptide to highly potent analogs of thapsigargin;(3) to evaluate pharmacology, toxicology and antitumor efficacy of these FAP-activated prodrugs in vivo. To accomplish Aim 1 we have generated recombinant FAP and generated a map of cleavage sites within recombinant collagen I. Peptides based on these cleavage sites will be evaluated as putative FAP substrates. In addition, we have generated a random phage substrate library to evaluate a more diverse collection of peptides as FAP substrates. In Aim 2, we will select the best of these FAP substrates to produce FAP activated thapsigargin prodrugs which will be characterized for FAP hydrolysis and stability in human and mouse plasma. In addition, the prodrugs will be tested for toxicity against a panel of FAP negative cancer cell lines and a FAP transfected line as a positive control. Prodrugs that are efficiently hydrolyzed by FAP, stable in plasma, and minimally toxic to FAP negative cell lines will be further evaluated in Aim 3 in vivo to determine efficacy against human cancer xenografts producing a range of stroma. These xenografts will be characterized for % stroma, for FAP production and enzymatic activity. Prior to efficacy studies, pharmacokinetic analysis and toxicology studies will be performed to determine optimal dosing regimen. The studies described in this proposal will define whether therapies that target the supporting structures (i.e. "stroma") within cancers rather than the cancer cells themselves can be effective therapy. The FAP- activated prodrug, therefore, could represent a new targeted therapy for a variety of human cancers.